1. Field of the Invention
The present invention relates to recombinant adeno-associated virus vectors for gene delivery and regulated tissue specific expression in at least one mammalian cell such that the expression of the gene is regulated in a tissue specific manner. The present invention also provides a method for using this adeno-associated virus vector for therapeutic purposes.
2. Brief Description of the Prior Art
It is well known that gene therapy of severe hemoglobinopathies requires high level regulated tissue specific expression of a transferred globin gene into hematopoietic stem cells and subsequent high level regulated tissue specific gene expression in maturing erythroid cells. In homozygous patients with such as for example, beta-thalassemia. deficient or absent beta-globin synthesis causes the production of poorly hemoglobinized-defective red cells resulting in severe anemia. Transfer and expression of a normal beta-globin gene, therefore, is highly effective in correcting the defect. In sickle cell anemia, the mutant hemoglobin is susceptible to polymerization resulting in altered red cell rheological and membrane properties leading to vaso-occlusion. An increase in production of one of the globin genes, fetal hemoglobin (hereinafter HbF), appears to ameliorate the severity of sickle cell disease. It will be understood by those skilled in the art that the regulated tissue specific production of normal globin gene products at normal levels in response to environmental stimuli in erythroid cells of a mammalian host with sickle cell anemia is therefore, therapeutic.
It is well known that most strategies for human gene therapy are based on the use of viral vectors for gene transfer. It has been shown that viral vectors capable of infecting virtually every cell in a target population are an efficient method of delivering nucleic acids into mammalian cells. Viral expression vectors have been developed using DNA viruses such as, for example, papovaviruses such as SV40, adenoviruses, herpes viruses, and poxviruses and RNA viruses, such as retroviruses. Generally, the most common used model vectors have been derived from murine and avian retroviruses. These retrovirus vectors utilize packaging cell lines which allow production of replication-defective vectors in the absence of wild-type retroviral helper. The defective retroviral vectors are able to infect and integrate into cells but cannot replicate. The ability to produce helper-free defective retrovirus using packaging cell lines protects against spread of the recombinant virus., and avoids possible dissemination,of recombinant virus-induced disease. It is known to use retroviral vectors to transfer the beta-globin gene into murine hematopoietic stem cells. Although the human beta-globin gene is expressed in most mammals, when transferred into murine hematopoietic stem cells it is only expressed to levels of about 1-2% of the mouse chromosomal beta-globin genes, a level too low to be of any therapeutic value. The disadvantage of the retrovirus packaging lines is that they have been shown to only produce low titers of virus or to produce high levels of wild-type retroviral helper. Another disadvantage is that while the retrovirus vectors can infect a broad class of cell types, cell replication and DNA synthesis are strictly required for provirus integration, therefore restricting efficient use of retroviral based vectors to replicating cells.
The recognition of human retroviruses over the past decade as the etiologic agent of Acquired Immunodeficiency Syndrome (hereinafter AIDS) and in some cases T-cell and hairy cell leukemia have created an awareness of the health risks potentially associated with the use of retrovirus vectors.
It is known by those skilled in the art that retrovirus vectors have resulted in tumors in non-human primate studies as a result of contaminating wild-type retrovirus generated from the packaging cell line. This again points to the unavoidable risk inherent in the retrovirus packaging system.
It is well known that adeno-associated virus (hereinafter AAV) is a human defective, human dependent parvovirus. AAV requires coinfection with another virus such as for example, an adenovirus or certain members of the herpes virus family, for productive infection in cultured cells. In a lytic infection, AAV DNA replicates as a 4.7 kilobase double-stranded molecule and is packaged into virions as linear single-strands of both polarities with no preference as to polarity. It has been shown that in the absence of coinfection with a helper virus, the AAV genome integrates via its termini into the host genome in a site specific manner and resides there in a latent state until the cell is infected with helper virus. When the cell is infected with the helper virus, the AAV DNA is rescued, replicates and establishes a normal productive (lytic) infection.
The single-stranded DNA genome of the human virus AAV-2 (a serotype of AAV) is 4675 base pairs in length and is flanked by inverted terminal repeated sequences of 145 base pairs each. The first 125 nucleotides from a palindromic sequence can form a xe2x80x9cTxe2x80x9d-shaped hairpin secondary structure and exist in either of two orientations with respect to the genome, designated flip or flop. It has been suggested that AAV replicates according to which the terminal hairpin of AAV is used as a primer for the initiation of DNA replication. It has been shown that the AAV sequences that are required in cis for packaging, integration/rescue, and replication of viral DNA are located within a 191 base pair sequence that includes the terminal repeat sequences. The viral DNA sequence displays two major open reading frames, one in the left half and the other in the right half of the conventional AAV map. At least two regions which, when mutated give rise to phenotypically distinct viruses in the AAV genome. The rep region, which occupies the conventional left half of the genome, encodes proteins that are required for viral replication and for viral rescue when the viral genome is integrated. The cap region which occupies the conventional right half of the genome encodes AAV capsid proteins. Mutants within these three regions are capable of DNA replication but do not produce virus. It is known that AAV contains three transcriptional promotersxe2x80x94p5, p19, and p40. Four rep proteins (rep 78, 68, 52 and 40) and three capsid proteins (VP-1, VP-2 and VP-3) are derived from alternate splicing of the RNA transcripts of these promoters. These three promoters regulate expression of the genes required for replication and encapsidation of the AAV genome.
It has been shown that the majority of the cis-acting regulatory elements required for regulated tissue specific globin gene expression flank the globin gene cluster or reside within the gene themselves. The majority of these cis-acting regulatory elements have been defined by DNase I hypersensitive sites (hereinafter HS) and are collectively termed the locus control region (hereinafter LCR). Four sites (5xe2x80x2 HS I-IV) have been shown to be located several kilobases 5xe2x80x2 to the epsilon-globin gene and one site (3xe2x80x2 HS VI) has been shown to be mapped 3xe2x80x2 to the beta-globin gene. The active elements of the LCR are encompassed within 300-400 base pairs of DNA found at each hypersensitive site and some have been narrowed down to about 30 nucleotides. It is known that the 5xe2x80x2 HS II, III and IV when linked to globin genes singly or in combination, substantially enhanced and regulated globin gene expression to a maximum level equivalent to that of endogenous globin genes in transfected erythroleukemia cells or transgenic animals. The past efforts by others to develop retroviral vectors containing globin genes with regulatory elements needed to achieve high level expression have been unsuccessful. These retroviral vectors have been shown to have limited ability to transfer genes that result in regulated tissue specific expression into primate pluripotent hematopoietic stem cells which are a necessary target for genetic therapy of hemoglobin disorders.
Several AAV vector systems have been designed which contain a recombinant plasmid capable of being packaged into AAV particles. These recombinant viruses function as vectors for maintenance or expression of a gene or a DNA sequence in eukaryotic cells when under control of an AAV or SV40 transcriptional promoter.
Hermonat and Muzyczka, 1984, Proc. Natl. Acad. Sci. 81:6466-6470, disclose production of a recombinant AAV (hereinafter rAAV) viral stock in which the neomycin resistant gene (hereinafter neo) was substituted for the AAV capsid region. Hermonat and Muzyczka discloses rAAV transduction of neomycin resistance into murine and human cell lines. Hermonat and Muzyczka state that the stable integrated viral vector can be rescued to produce replicating rAAV sequences after superinfection with adenovirus and wild-type AAV.
Tratschin et al., 1984, Mol. Cell Biol. 4:2072-2081, disclose a rAAV that expresses the chloramphenicol acetyltransferase (hereinafter CAT) gene in human cells under the AAV p40 promoter.
Laface et al., 1988, Virology, 162:483-486, mentions possible gene transfer into hematopoietic progenitor cells mediated by an AAV vector. However, transduction efficiency was extremely low and was determined solely from the number of geneticin-resistant bone marrow colonies. Also, it was possible that this early generation rAAV preparation was contaminated with wild-type AAV virions which decreased rAAV transduction efficiency.
Wondisford et al., 1988, Mol. Endocrinol. 2:32-39, discloses co-transfected cells with two different recombinant AAV vectors each encoding a subunit of human thyrotropin. Wondisford et al. states that expression of biologically active thyrotropin was observed.
In spite of these prior art disclosures, there remains a very real and substantial need for a recombinant adeno-associated virus vector capable of delivering and expressing at least one mammalian gene into a genome of a mammalian host cell such that the expression of the gene is regulated in a tissue specific manner by cis-acting regulating and promoter elements and a method of using the recombinant adeno-associated virus vector for delivering and expressing genes into the cells of a mammalian host in vitro as well as in vivo for therapeutic purposes. More specifically, there is a need for a recombinant adeno-associated virus vector for delivery and expression in a regulated tissue specific manner of globin gene nucleotide sequences and cis-acting elements in erythroid cells of a mammalian host wherein the globin gene is regulated in a tissue specific manner by cis-acting regulatory and promoter elements. There is a need for such vectors capable of transferring coding sequences of a gene under the control of the native genomic transcriptional regulatory elements of that gene for achieving tissue specific, regulated expression of the transferred gene.
The present invention has met the hereinbefore described needs. The present invention provides a recombinant adeno-associated virus vector capable of delivering and expressing at least one mammalian gene into a genome of a mammalian host cell. The recombinant adeno-associated virus vector of this invention includes at least one mammalian gene, cis-acting regulatory and promoter elements of the gene and an adeno-associated virus such that the expression of the gene is regulated in a tissue specific manner by the cis-acting regulatory and promoter elements. More specifically, the mammalian gene is a human gene wherein the cis-acting regulatory elements are human cis-acting regulatory elements and wherein said promoter elements are human promoter elements.
Another embodiment of this invention provides for the recombinant adeno-associated virus vector of this invention wherein the mammalian gene is a hybrid gene.
A further embodiment of this invention provides for the recombinant adeno-associated virus vector of this invention wherein the cis-acting regulatory and promoter elements are of a globin gene cluster. Preferably, the mammalian gene is a human gene and the cis-acting regulatory and promoter elements are of a human globin gene cluster. This human globin gene cluster includes the cis-acting regulatory and promoter elements located between hypersensitive sites IV and VI.
Another embodiment of this invention provides for the recombinant adeno-associated virus vector of this invention wherein the mammalian gene is a globin gene. More specifically, the globin gene is a hybrid gene.
Another embodiment of this invention provides for the recombinant adeno-associated virus vector of this invention wherein the globin gene is at least one gene selected from the gene cluster group that includes epsilon-globin, Agamma-globin, Ggamma-globin, delta-globin, beta-globin and combinations thereof. This globin gene is linked to at least one hypersensitive site wherein the hypersensitive site is located within the globin gene cluster. More specifically, this globin gene is linked to at least one hypersensitive site wherein the hypersensitive site is located within a locus control region.
A specific embodiment of a rAAV vector of the present invention is the rAAV vector, pAAV/HSII Axcex3*/Neo, as depicted in FIG. 1 and disclosed in Example I.
It is within the scope of the present invention to construct various rAAV constructs comprising HS-based promoter/enhancer fragments fused to the above-mentioned globin genes or gene fragments for transfer to target cell types disclosed throughout this specification.
The present invention is in no way limited to utilizing only rAAV recombinant vectors which comprise HS/globin hybrid constructs for targeting to the appropriate cell type. Instead, the present invention is directed, as noted in the Field of the Invention, to rAAV vectors which are delivered to and expressed in a tissue specific manner within the mammalian host.
To this end, the present invention is also related to non-HS/globin constructs for treating a mammalian host, preferably a human, having a hemoglobinopathy, a blood borne disorder, genetic disease or an acquired disease. This concept is shown in one fashion by the data of Example II, which shows expression of rAAV-xcex2-gal constructs in primary hematopbietic progenitors. A preferred embodiment of this portion of the present invention, included as an example but not a limitation, is the plasmid rAAV/FACC/NeoR, which is utilized to direct phenotypic correction of Fanconi anemia in hematopoietic cells.
A further embodiment of this invention provides for the adeno-associated virus vector of this invention wherein the mammalian host cell includes a bone marrow stem cell and derivatives thereof and an immune cell such as a human hematopoietic stem cell, a human myeloid progenitor cell and a human erythroid progenitor cell. More specifically, the bone marrow stem cell is at least one cell that includes a non-human bone marrow stem cell and a human bone marrow stem cell, and the bone marrow stem cell derivative is at least one cell that includes a derivative of the non-human bone marrow stem cell and a derivative of the human bone marrow stem cell.
Another embodiment of this invention provides a cell product of the recombinant adeno-associated virus vector of this invention.
Further, this invention provides a method of using the recombinant adeno-associated virus vector of this invention that is capable of delivering and expressing in a regulated tissue specific manner at least one mammalian gene into a genome of a mammalian host cell for therapeutic purposes. More specifically, this method includes treating the mammalian host having a hemoglobinopathy, a blood borne disorder, genetic disease or an acquired disease.
Another specific embodiment of the present invention is exemplified by the rAAV vector, JM24/HS432Axcex3*. Example IV shows efficient transfer, integration and expression of the globin gene expressed by this rAAV vector in primary erythroid cells. This rAAV construct employs the HSIV, HSIII and HSII regions of the LCR discussed within this specification. These rAAV constructs exemplify a running theme of the present invention: rAAV vectors disclosed in the specification are useful in gene therapy applications in general and in treatment of various hemoglobinopathies in particular. The effectiveness of the rAAV vectors of the present invention is limited only by an ability to transfer a rAAV hybrid promoter/gene cassette to the cell type of interest so as to promote tissue specific expression within the target cell, in this case, primary human hematopoietic cells.
To this end, the present invention also relates to non-HS/globin constructs for treating a mammalian host, preferably a human, having a hemoglobinpathy, a blood-borne disorder, genetic disease or acquired disease. A specific and preferred embodiment of this portion of the present invention, included as an example but not as a limitation, is the plasmid rAAV/FACC/NeoR, which is shown in Example III to direct phenotypic correction of Fanconi anemia in hematopoietic cells.
It is also an embodiment of the present invention to treat additional human diseases or disorders by affecting tissue specific expression of rAAV-based therapeutic DNA sequences. As examples, but not forwarded as limitations, the rAAV vectors of the present invention may be utilized for liver specific expression of Factor IX for treatment of hemophilia, expression of CTRF in the lung for treatment of cystic fibrosis, and expression of tyrosine hydrolase in brain tissue for treating Parkinson""s disease.
It is an object of the present invention to provide a recombinant adeno-associated virus vector capable of delivering and expressing at least one mammalian gene into a genome of a mammalian host cell for effecting genetic therapy such that the expression of the gene is regulated in a tissue specific manner.
It is an object of the present invention to provide a recombinant adeno-associated virus vector that is capable of delivering and expressing at least one mammalian gene into a genome of a mammalian host wherein the mammalian gene is a hybrid gene, and wherein the expression of this gene is regulated in a tissue specific manner.
It is another object of the present invention to provide a recombinant adeno-associated virus vector that is capable of delivering and expressing at least one human gene into a genome of a mammalian host cell such that the expression is regulated in a tissue specific manner.
It is a further object of the present invention to provide a recombinant adeno-associated virus vector capable of delivering and expressing at least one human globin gene into a genome of a mammalian host such that the expression of the gene is regulated in a tissue specific manner by cis-acting regulator and promoter elements of a human globin gene cluster that includes cis-acting regulatory and promoter elements located between hypersensitive sites IV and VI.
It is an object of the present invention to provide a method for using the recombinant adeno-associated virus vector of this invention for delivering and expressing at least one human gene into a genome of a human host cell for therapeutic purposes such that the expression of the human gene is regulated in a tissue specific manner.
It is a further object of this invention to provide a method for using the recombinant adeno-associated vector of this invention for delivering and expressing at least one globin gene into a genome of a mammalian host cell including employing this method for treating the mammalian host having a hemoglobinopathy, a blood borne disorder, a genetic disease or an acquired disease, in which tissue specific and regulated expression of the delivered gene is desirable.
It is an object of the present invention to utilize the rAAV vector constructs of the present invention to affect therapeutic relief or prophylactic intervention to various human diseases or disorders, both blood-borne and non blood-borne; which are amenable to tissue specific rAAV transduction and target specific expression of a gene or gene fragment which will provide said relief or intervention.